This invention relates to the field of producing foreign proteins in host bacteria using recombinant techniques. More specifically, the invention relates to novel control sequences which regulate the expression of a desired gene and thus to production of its encoded protein in response to factors under the control of the experimenter. The invention is applicable to a wide range of prokaryotic hosts.
It is now understood quite clearly that there is more to the successful use of recombinant techniques to produce desired proteins than merely inserting the appropriate gene into an expression vector and transforming a suitable host. Not only must the expression system be recognized by the host cell, but the timing of the expression must be regulated to insure that the protein is produced at high levels only when the cell can best tolerate the amounts of foreign protein. Foreign protein genes are often expressed at levels that produce protein in much greater amounts than those of any endogenous protein. If such large amounts of protein are the goal, they may be lethal, and expression is often best separated from growth phase. Alternatively, it may be desirable to regulate the specific level of protein production so as to optimize its functionality in the context of other cellular events. Accordingly, workers in this field have employed promoters which are susceptible to control by repressor binding to operator sequences either contained within the promoter sequence or slightly downstream.
Microorganisms themselves utilize several control mechanisms for regulating the level of protein production. In some organisms and for some proteins, control is exerted at the translational level by direct inhibition or stimulation of the rate of protein synthesis at the ribosome or by stabilization or destabilization of mRNA. This translational approach at present does not easily lend itself to fine-tuned voluntary control by the experimenter, and has not been used to obtain the desired objectives of recombinant technology such as high levels of protein production. At least two control strategies have been described which are used by microorganisms at the level of transcription. One involves "sigma factors" which are produced by microorganisms at various stages of their life cycles, and which bind to RNA polymerase to render it more, or less, suitable for particular promoter sites in the DNA sequences to be transcribed. This method, like translational control, is not presently employed as a means to effect external control of expression, because it does not permit the desired level of controllability.
A second transcriptional strategy employs an "operator"--i.e., a sequence in the operon proximal to the promoter either included within the promoter itself or somewhat downstream, to which a repressor molecule is bound when the transcription is to be shut off. The repressor is removed from the operator in response to a depletion in its total effective amount, often obtained by supplying to the cell an inductor which inactivates the repressor protein. It is this strategy which has been co-opted by biotechnologists to effect their own control over expression. For example, see Sninsky, J. J., et al., Gene, 16: 275 (1981). However, heretofore, this approach has been available only in E. coli and closely related gram negative hosts, since it is only in these systems that the operator/repressor mechanism has been described. Furthermore, such control systems have not been constructed in a form so that they can function in conjunction with any desired gene for expression in other hosts besides the natural host for the control system. The ability to so function renders the system "portable."
The present invention provides a portable control system which comprises a promoter/operator sequence wherein the operator is potentially under the control of a repressor, plus the coding sequence for this repressor also operably linked to a suitable promoter. Such a compilation of sequences can be utilized in a large variety of hosts including hosts which are not known to employ such control systems endogenously. Thus a control system is provided which can be inserted into plasmid vectors or into the genome of the desired host organism so as to provide an inducible transcription control for a gene sequence operably linked to it. This is a suitable regulation system for genes encoding desired proteins which is easily manipulated and controlled, both in traditional E. coli hosts and more importantly, in less traditional, gram positive hosts. These latter hosts within their own complement of genetic and plasmid material either lack this system of controlling gene expression altogether or such systems of regulation are not yet known to be associated with them.